Smart-seq2 Single Nucleus Multi-Sample Count Matrix Overview

The Smart-seq2 Single Nucleus Multi-Sample (Multi-snSS2) pipeline's default count matrix output is a Loom file, an HDF5 file generated using Loompy v.3.0.6. It contains the raw cell-by-gene intron and exon counts.

The matrix also contains multiple metrics for both individual cells (the columns of the matrix; Table 2) and individual genes (the rows of the matrix; Table 3).

Additional details for each metric are provided in the JAVA source code for Picard's AlignmentSummaryMetrics, GcBiasSummaryMetrics, and DuplicationMetrics.

Table 1. Global attributes

The global attributes in the Loom apply to the whole file, not any specific part.

Attribute	Details
CreationDate	Date the Loom file was created.
LOOM_SPEC_VERSION	Loom file spec version used during creation of the Loom file.
batch_id	The batch_id provided to the pipeline as input.
pipeline_version	Version of the Multi-snSS2 pipeline used to generate the Loom file.

Table 2. Column attributes (cell metrics)

The cell metrics below are computed using Picard, with the exception of CellID, cell_names, and input_id which are provided to the pipeline as input.

Cell Metrics	Tool	Details
ACCUMULATION_LEVEL	CollectMultipleMetrics	Level of metric accumulation; set to ALL_READS using the --METRIC_ACCUMULATION_LEVEL argument.
ALIGNED_READS	CollectGcBiasMetrics	Total number of aligned reads produced in a run.
AT_DROPOUT	CollectGcBiasMetrics	Percentage of misaligned reads with GC content below 50%.
AVG_POS_3PRIME_SOFTCLIP_LENGTH.FIRST_OF_PAIR	CollectAlignmentSummaryMetrics	Average length of soft-clipped bases at the 3' end of the first reads.
AVG_POS_3PRIME_SOFTCLIP_LENGTH.PAIR	CollectAlignmentSummaryMetrics	Average length of soft-clipped bases at the 3' end of all reads.
AVG_POS_3PRIME_SOFTCLIP_LENGTH.SECOND_OF_PAIR	CollectAlignmentSummaryMetrics	Average length of soft-clipped bases at the 3' end of the second reads.
BAD_CYCLES.FIRST_OF_PAIR	CollectAlignmentSummaryMetrics	Number of cycles with combined no-call and mismatch rates greater than or equal to 80% for the first reads.
BAD_CYCLES.PAIR	CollectAlignmentSummaryMetrics	Number of cycles with combined no-call and mismatch rates greater than or equal to 80% for all reads.
BAD_CYCLES.SECOND_OF_PAIR	CollectAlignmentSummaryMetrics	Number of cycles with combined no-call and mismatch rates greater than or equal to 80% for the second reads.
CellID	warp-tools	Unique identifier for each cell provided to the pipeline as input_ids; identical to cell_names and input_id.
ESTIMATED_LIBRARY_SIZE	MarkDuplicates	Estimated number of unique molecules in the library based on paired-end duplication.
GC_DROPOUT	CollectGcBiasMetrics	Percentage of misaligned reads with GC content above 50%.
GC_NC_0_19	CollectGcBiasMetrics	Normalized coverage over reads with GC content from 0 - 19%.
GC_NC_20_39	CollectGcBiasMetrics	Normalized coverage over reads with GC content from 20 - 39%.
GC_NC_40_59	CollectGcBiasMetrics	Normalized coverage over reads with GC content from 40 - 59%.
GC_NC_60_79	CollectGcBiasMetrics	Normalized coverage over reads with GC content from 60 - 79%.
GC_NC_80_100	CollectGcBiasMetrics	Normalized coverage over reads with GC content from 80 - 100%.
MAD_READ_LENGTH.FIRST_OF_PAIR	CollectAlignmentSummaryMetrics	Median absolute deviation of the lengths of forward reads.
MAD_READ_LENGTH.PAIR	CollectAlignmentSummaryMetrics	Median absolute deviation of the lengths of all reads.
MAD_READ_LENGTH.SECOND_OF_PAIR	CollectAlignmentSummaryMetrics	Median absolute deviation of the lengths of reverse reads.
MAX_READ_LENGTH.FIRST_OF_PAIR	CollectAlignmentSummaryMetrics	Maximum length of forward reads.
MAX_READ_LENGTH.PAIR	CollectAlignmentSummaryMetrics	Maximum length of all reads.
MAX_READ_LENGTH.SECOND_OF_PAIR	CollectAlignmentSummaryMetrics	Maximum length of reverse reads.
MEAN_READ_LENGTH.FIRST_OF_PAIR	CollectAlignmentSummaryMetrics	Mean length of forward reads.
MEAN_READ_LENGTH.PAIR	CollectAlignmentSummaryMetrics	Mean length of all reads.
MEAN_READ_LENGTH.SECOND_OF_PAIR	CollectAlignmentSummaryMetrics	Mean length of reverse reads.
MEDIAN_READ_LENGTH.FIRST_OF_PAIR	CollectAlignmentSummaryMetrics	Median length of forward reads.
MEDIAN_READ_LENGTH.PAIR	CollectAlignmentSummaryMetrics	Median length of all reads.
MEDIAN_READ_LENGTH.SECOND_OF_PAIR	CollectAlignmentSummaryMetrics	Median length of reverse reads.
MIN_READ_LENGTH.FIRST_OF_PAIR	CollectAlignmentSummaryMetrics	Minimum length of forward reads.
MIN_READ_LENGTH.PAIR	CollectAlignmentSummaryMetrics	Minimum length of all reads.
MIN_READ_LENGTH.SECOND_OF_PAIR	CollectAlignmentSummaryMetrics	Minimum length of reverse reads.
PCT_ADAPTER.FIRST_OF_PAIR	CollectAlignmentSummaryMetrics	Fraction of pass-filter forward reads that are unaligned or aligned with a mapping quality of 0 and match to a known adapter sequence from the start of the read.
PCT_ADAPTER.PAIR	CollectAlignmentSummaryMetrics	Fraction of all pass-filter reads that are unaligned or aligned with a mapping quality of 0 and match to a known adapter sequence from the start of the read.
PCT_ADAPTER.SECOND_OF_PAIR	CollectAlignmentSummaryMetrics	Fraction of pass-filter reverse reads that are unaligned or aligned with a mapping quality of 0 and match to a known adapter sequence from the start of the read.
PCT_CHIMERAS.FIRST_OF_PAIR	CollectAlignmentSummaryMetrics	Fraction of forward reads where the insert is larger than 100 kb or the ends of the pair map to different chromosomes.
PCT_CHIMERAS.PAIR	CollectAlignmentSummaryMetrics	Fraction of all reads where the insert is larger than 100 kb or the ends of the pair map to different chromosomes.
PCT_CHIMERAS.SECOND_OF_PAIR	CollectAlignmentSummaryMetrics	Fraction of reverse reads where the insert is larger than 100 kb or the ends of the pair map to different chromosomes.
PCT_HARDCLIP.FIRST_OF_PAIR	CollectAlignmentSummaryMetrics	Fraction of pass-filter bases that are hard-clipped from aligned, forward reads.
PCT_HARDCLIP.PAIR	CollectAlignmentSummaryMetrics	Fraction of pass-filter bases that are hard-clipped from all aligned reads.
PCT_HARDCLIP.SECOND_OF_PAIR	CollectAlignmentSummaryMetrics	Fraction of pass-filter bases that are hard-clipped from aligned, reverse reads.
PCT_PF_READS.FIRST_OF_PAIR	CollectAlignmentSummaryMetrics	Fraction of forward reads that pass vendor check (pass-filter).
PCT_PF_READS.PAIR	CollectAlignmentSummaryMetrics	Fraction of reads that pass vendor check (pass-filter).
PCT_PF_READS.SECOND_OF_PAIR	CollectAlignmentSummaryMetrics	Fraction of reverse reads that pass vendor check (pass-filter).
PCT_PF_READS_ALIGNED.FIRST_OF_PAIR	CollectAlignmentSummaryMetrics	Fraction of pass-filter forward reads that are aligned.
PCT_PF_READS_ALIGNED.PAIR	CollectAlignmentSummaryMetrics	Fraction of all pass-filter reads that are aligned.
PCT_PF_READS_ALIGNED.SECOND_OF_PAIR	CollectAlignmentSummaryMetrics	Fraction of pass-filter reverse reads that are aligned.
PCT_PF_READS_IMPROPER_PAIRS.FIRST_OF_PAIR	CollectAlignmentSummaryMetrics	Fraction of forward reads not properly aligned in pairs.
PCT_PF_READS_IMPROPER_PAIRS.PAIR	CollectAlignmentSummaryMetrics	Fraction of reads not properly aligned in pairs.
PCT_PF_READS_IMPROPER_PAIRS.SECOND_OF_PAIR	CollectAlignmentSummaryMetrics	Fraction of reverse reads not properly aligned in pairs.
PCT_READS_ALIGNED_IN_PAIRS.FIRST_OF_PAIR	CollectAlignmentSummaryMetrics	Fraction of forward reads properly aligned in pairs.
PCT_READS_ALIGNED_IN_PAIRS.PAIR	CollectAlignmentSummaryMetrics	Fraction of reads properly aligned in pairs.
PCT_READS_ALIGNED_IN_PAIRS.SECOND_OF_PAIR	CollectAlignmentSummaryMetrics	Fraction of reverse reads properly aligned in pairs.
PCT_SOFTCLIP.FIRST_OF_PAIR	CollectAlignmentSummaryMetrics	Fraction of pass-filter bases that are soft-clipped from aligned forward reads.
PCT_SOFTCLIP.PAIR	CollectAlignmentSummaryMetrics	Fraction of pass-filter bases that are soft-clipped from all aligned reads.
PCT_SOFTCLIP.SECOND_OF_PAIR	CollectAlignmentSummaryMetrics	Fraction of pass-filter bases that are soft-clipped from aligned reverse reads.
PERCENT_DUPLICATION	MarkDuplicates	Fraction of mapped sequence marked as duplicate.
PF_ALIGNED_BASES.FIRST_OF_PAIR	CollectAlignmentSummaryMetrics	Total number of aligned bases in pass-filter forward reads.
PF_ALIGNED_BASES.PAIR	CollectAlignmentSummaryMetrics	Total number of aligned bases in all pass-filter reads.
PF_ALIGNED_BASES.SECOND_OF_PAIR	CollectAlignmentSummaryMetrics	Total number of aligned bases in pass-filter reverse reads.
PF_HQ_ALIGNED_BASES.FIRST_OF_PAIR	CollectAlignmentSummaryMetrics	Number of bases aligned to the reference sequence in forward reads with high mapping quality.
PF_HQ_ALIGNED_BASES.PAIR	CollectAlignmentSummaryMetrics	Number of bases aligned to the reference sequence in all reads with high mapping quality.
PF_HQ_ALIGNED_BASES.SECOND_OF_PAIR	CollectAlignmentSummaryMetrics	Number of bases aligned to the reference sequence in reverse reads with high mapping quality.
PF_HQ_ALIGNED_Q20_BASES.FIRST_OF_PAIR	CollectAlignmentSummaryMetrics	Subset of PF_HQ_ALIGNED_BASES.FIRST_OF_PAIR with a base call quality of at least 20.
PF_HQ_ALIGNED_Q20_BASES.PAIR	CollectAlignmentSummaryMetrics	Subset of PF_HQ_ALIGNED_BASES.PAIR with a base call quality of at least 20.
PF_HQ_ALIGNED_Q20_BASES.SECOND_OF_PAIR	CollectAlignmentSummaryMetrics	Subset of PF_HQ_ALIGNED_BASES.SECOND_OF_PAIR with a base call quality of at least 20.
PF_HQ_ALIGNED_READS.FIRST_OF_PAIR	CollectAlignmentSummaryMetrics	Number of pass-filter forward reads aligned with a mapping quality of at least 20.
PF_HQ_ALIGNED_READS.PAIR	CollectAlignmentSummaryMetrics	Number of all pass-filter reads aligned with a mapping quality of at least 20.
PF_HQ_ALIGNED_READS.SECOND_OF_PAIR	CollectAlignmentSummaryMetrics	Number of pass-filter reverse reads aligned with a mapping quality of at least 20.
PF_HQ_ERROR_RATE.FIRST_OF_PAIR	CollectAlignmentSummaryMetrics	Fraction of bases in pass-filter, high-quality forward reads that do not match the reference.
PF_HQ_ERROR_RATE.PAIR	CollectAlignmentSummaryMetrics	Fraction of bases in all pass-filter, high-quality reads that do not match the reference.
PF_HQ_ERROR_RATE.SECOND_OF_PAIR	CollectAlignmentSummaryMetrics	Fraction of bases in pass-filter, high-quality reverse reads that do not match the reference.
PF_HQ_MEDIAN_MISMATCHES.FIRST_OF_PAIR	CollectAlignmentSummaryMetrics	Median number of mismatches in high-quality forward reads.
PF_HQ_MEDIAN_MISMATCHES.PAIR	CollectAlignmentSummaryMetrics	Median number of mismatches in all high-quality reads.
PF_HQ_MEDIAN_MISMATCHES.SECOND_OF_PAIR	CollectAlignmentSummaryMetrics	Median number of mismatches in high-quality reverse reads.
PF_INDEL_RATE.FIRST_OF_PAIR	CollectAlignmentSummaryMetrics	Number of insertion and deletion events per 100 aligned bases in forward reads.
PF_INDEL_RATE.PAIR	CollectAlignmentSummaryMetrics	Number of insertion and deletion events per 100 aligned bases in all reads.
PF_INDEL_RATE.SECOND_OF_PAIR	CollectAlignmentSummaryMetrics	Number of insertion and deletion events per 100 aligned bases in reverse reads.
PF_MISMATCH_RATE.FIRST_OF_PAIR	CollectAlignmentSummaryMetrics	Rate of base mismatching for all aligned bases in forward reads.
PF_MISMATCH_RATE.PAIR	CollectAlignmentSummaryMetrics	Rate of base mismatching for all aligned bases in all reads.
PF_MISMATCH_RATE.SECOND_OF_PAIR	CollectAlignmentSummaryMetrics	Rate of base mismatching for all aligned bases in reverse reads.
PF_NOISE_READS.FIRST_OF_PAIR	CollectAlignmentSummaryMetrics	Number of pass-filter forward reads marked as noise.
PF_NOISE_READS.PAIR	CollectAlignmentSummaryMetrics	Number of all pass-filter reads marked as noise.
PF_NOISE_READS.SECOND_OF_PAIR	CollectAlignmentSummaryMetrics	Number of pass-filter reverse reads marked as noise.
PF_READS.FIRST_OF_PAIR	CollectAlignmentSummaryMetrics	Number of forward reads that pass vendor check (pass-filter).
PF_READS.PAIR	CollectAlignmentSummaryMetrics	Number of reads that pass vendor check (pass-filter).
PF_READS.SECOND_OF_PAIR	CollectAlignmentSummaryMetrics	Number of reverse reads that pass vendor check (pass-filter).
PF_READS_ALIGNED.FIRST_OF_PAIR	CollectAlignmentSummaryMetrics	Number of pass-filter forward reads that are aligned.
PF_READS_ALIGNED.PAIR	CollectAlignmentSummaryMetrics	Number of pass-filter reads that are aligned.
PF_READS_ALIGNED.SECOND_OF_PAIR	CollectAlignmentSummaryMetrics	Number of pass-filter reverse reads that are aligned.
PF_READS_IMPROPER_PAIRS.FIRST_OF_PAIR	CollectAlignmentSummaryMetrics	Number of forward reads not properly aligned in pairs.
PF_READS_IMPROPER_PAIRS.PAIR	CollectAlignmentSummaryMetrics	Number of reads not properly aligned in pairs.
PF_READS_IMPROPER_PAIRS.SECOND_OF_PAIR	CollectAlignmentSummaryMetrics	Number of reverse reads not properly aligned in pairs.
READS_ALIGNED_IN_PAIRS.FIRST_OF_PAIR	CollectAlignmentSummaryMetrics	Number of forward reads properly aligned in pairs.
READS_ALIGNED_IN_PAIRS.PAIR	CollectAlignmentSummaryMetrics	Number of reads properly aligned in pairs.
READS_ALIGNED_IN_PAIRS.SECOND_OF_PAIR	CollectAlignmentSummaryMetrics	Number of reverse reads properly aligned in pairs.
READS_USED	CollectGcBiasMetrics	String describing whether duplicates are included in metrics produced by CollectGcBiasMetrics; the pipeline removes duplicates before metrics are calculated.
READ_PAIRS_EXAMINED	MarkDuplicates	Number of mapped read pairs examined by MarkDuplicates.
READ_PAIR_DUPLICATES	MarkDuplicates	Number of read pairs marked as duplicates.
READ_PAIR_OPTICAL_DUPLICATES	MarkDuplicates	Number of read pairs duplicates caused by optical duplication.
SD_READ_LENGTH.FIRST_OF_PAIR	CollectAlignmentSummaryMetrics	Standard deviation of forward read lengths.
SD_READ_LENGTH.PAIR	CollectAlignmentSummaryMetrics	Standard deviation of read lengths.
SD_READ_LENGTH.SECOND_OF_PAIR	CollectAlignmentSummaryMetrics	Standard deviation of reverse read lengths.
SECONDARY_OR_SUPPLEMENTARY_RDS	MarkDuplicates	Number of secondary or supplemetary reads.
STRAND_BALANCE.FIRST_OF_PAIR	CollectAlignmentSummaryMetrics	Number of pass-filter forward reads aligned divided by the total number of pass-filter reads aligned.
STRAND_BALANCE.PAIR	CollectAlignmentSummaryMetrics	Average strand balance of forward and reverse reads.
STRAND_BALANCE.SECOND_OF_PAIR	CollectAlignmentSummaryMetrics	Number of pass-filter reverse reads aligned divided by the total number of pass-filter reads aligned.
TOTAL_CLUSTERS	CollectGcBiasMetrics	Total number of reads after filtering used in GC bias calculation.
TOTAL_READS.FIRST_OF_PAIR	CollectAlignmentSummaryMetrics	Total number of forward reads.
TOTAL_READS.PAIR	CollectAlignmentSummaryMetrics	Total number of reads.
TOTAL_READS.SECOND_OF_PAIR	CollectAlignmentSummaryMetrics	Total number of reverse reads.
UNMAPPED_READS	MarkDuplicates	Number of unmapped reads examined by MarkDuplicates.
UNPAIRED_READS_EXAMINED	MarkDuplicates	Number of mapped reads without a mapped mate pair examined by MarkDuplicates.
UNPAIRED_READ_DUPLICATES	MarkDuplicates	Number of fragments marked as duplicates.
WINDOW_SIZE	CollectGcBiasMetrics	Genomic window size used in GC bias calculation.
cell_names	warp-tools	Unique identifier for each cell provided to the pipeline as input_ids; identical to Cell_ID and input_id.
input_id	warp-tools	Unique identifier for each cell provided to the pipeline as input_ids; identical to Cell_ID and cell_names.

Table 3. Row attributes (gene metrics)

Gene Metrics	Tool	Details
Gene	GENCODE GTF	The unique gene_ids provided in the GENCODE GTF; identical to the ensembl_ids attribute.
ensembl_ids	GENCODE GTF	The unique gene_ids provided in the GENCODE GTF; identical to the Gene attribute.
exon_lengths	warp-tools	The length in base pairs of the exons corresponding to this entity.
gene_names	GENCODE GTF	The unique gene_name provided in the GENCODE GTF.
intron_lengths	warp-tools	The length in base pairs of the introns corresponding to this entity.